Large‐Scale Preparation and Concentration of Retrovirus Stocks

Constance Cepko1

1 Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 9.12
DOI:  10.1002/0471142727.mb0912s37
Online Posting Date:  May, 2001
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Abstract

For some applications, such as infection of cells in vivo, it is necessary to concentrate retrovirus stocks in order to increase their titer. Because viruses are macromolecular structures, they can be concentrated fairly easily by a relatively short centrifugation step. This unit provides protocols in which the viral particles are either pelleted or centrifuged onto a sucrose density step gradient. An alternate protocol details how virions can be precipitated using polyethylene glycol or ammonium sulfate, and the resulting precipitate collected by centrifugation. After resuspension of the precipitates, the virions can be used directly, or further purified either by sedimentation onto sucrose density gradients, or by column filtration as described here. Perhaps most simply of all, an alternate procedure describes how small volumes of virus stock can be concentrated by centrifugation through a filter that allows only small molecules to pass.

     
 
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Table of Contents

  • Basic Protocol 1: Preparation of Virus Stock and Concentration by Centrifugation
  • Alternate Protocol 1: Concentration by PEG Precipitation and Chromatography
  • Alternate Protocol 2: Concentration Using Molecular‐Weight‐Cutoff Filters
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Preparation of Virus Stock and Concentration by Centrifugation

  Materials
  • Identified high‐titer Ψ2 producer cells, 50% to 90% confluent (unit 9.10)
  • Complete DMEM containing 10% calf serum (DMEM‐10, appendix 3F, prepared with calf serum instead of FBS)
  • NIH 3T3 cells
  • 800 µg/ml polybrene
  • 0.45‐µm filters for large volumes (e.g., Nalgene 115‐ml or 500‐ml filters)
  • Beckman JA‐14 rotor with 250‐ml centrifuge bottles (or equivalent) if concentrating large volumes or Beckman SW‐27 or SW‐41Ti rotor (or equivalent) if concentrating small volumes
NOTE: All incubations involving tissue culture cells should be performed in a humidified incubator 37°C, 5% CO 2 unless otherwise noted.

Alternate Protocol 1: Concentration by PEG Precipitation and Chromatography

  Additional Materials
  • 5 M NaCl, filter sterilized
  • Polyethylene glycol (PEG) 6000, filter sterilized
  • recipeNTE buffer (see recipe)
  • Sepharose CL‐4B or CL‐2B (Pharmacia)
  • Savant high‐speed centrifuge or Beckman SW‐41Ti rotor
  • Additional equipment for preparation of Sepharose column (unit 5.6)
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Figures

Videos

Literature Cited

Literature Cited
   Aboud, M., Wolfson, M., Hassan, Y., and Huleihel, M. 1982. Rapid purification of extracellular and intracellular Moloney murine leukemia virus. Arch. Virol. 71:185‐195.
   Stoker, A.W., Hatier, C., and Bissell, M.J. 1990. The embryonic environment strongly attenuates v‐src oncogenesis in mesenchymal and epithelial tissues, but not in endothelia. J. Cell Biol. 111:217‐228.
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